0 Q&A 43 Views Dec 5, 2022

Pathogen invasion of the central nervous system (CNS) is an important cause of infection-related mortality worldwide and can lead to severe neurological sequelae. To gain access to the CNS cells, pathogens have to overcome the blood–brain barrier (BBB), a protective fence from blood-borne factors. To study host–pathogen interactions, a number of cell culture and animal models were developed. However, in vitro models do not recapitulate the 3D architecture of the BBB and CNS tissue, and in vivo mammalian models present cellular and technical complexities as well as ethical issues, rendering systematic and genetic approaches difficult. Here, we present a two-pronged methodology allowing and validating the use of Drosophila larvae as a model system to decipher the mechanisms of infection in a developing CNS. First, an ex vivo protocol based on whole CNS explants serves as a fast and versatile screening platform, permitting the investigation of molecular and cellular mechanisms contributing to the crossing of the BBB and consequences of infection on the CNS. Then, an in vivo CNS infection protocol through direct pathogen microinjection into the fly circulatory system evaluates the impact of systemic parameters, including the contribution of circulating immune cells to CNS infection, and assesses infection pathogenicity at the whole host level. These combined complementary approaches identify mechanisms of BBB crossing and responses of a diversity of CNS cells contributing to infection, as well as novel virulence factors of the pathogen.

Graphical abstract

Procedures flowchart.
Mammalian neurotropic pathogens could be tested in two Drosophila central nervous system (CNS) infection setups (ex vivo and in vivo) for their ability to: (1) invade the CNS (pathogen quantifications), (2) disturb blood–brain barrier permeability, (3) affect CNS host cell behaviour (gene expression), and (4) alter host viability.

0 Q&A 360 Views Nov 20, 2022

The study of haloarchaea provides an opportunity to expand understanding of the mechanisms used by extremophiles to thrive in and respond to harsh environments, including hypersaline and oxidative stress conditions. A common strategy used to investigate molecular mechanisms of stress response involves the deletion and/or site-directed mutagenesis of genes identified through omics studies followed by a comparison of the mutant and wild-type strains for phenotypic differences. The experimental methods used to monitor these differences must be controlled and reproducible. Current methods to examine recovery of halophilic archaea from extreme stress are complicated by extended incubation times, nutrients not typically encountered in the environment, and other related limitations. Here we describe a method for assessing the function of genes during hypochlorite stress in the halophilic archaeon Haloferax volcanii that overcomes these types of limitations. The method was found reproducible and informative in identifying genes needed for H. volcanii to recover from hypochlorite stress.

0 Q&A 716 Views Nov 20, 2022

The study and use of decellularized extracellular matrix (dECM) in tissue engineering, regenerative medicine, and pathophysiology have become more prevalent in recent years. To obtain dECM, numerous decellularization procedures have been developed for the entire organ or tissue blocks, employing either perfusion of decellularizing agents through the tissue’s vessels or submersion of large sections in decellularizing solutions. However, none of these protocols are suitable for thin tissue slices (less than 100 µm) or allow side-by-side analysis of native and dECM consecutive tissue slices. Here, we present a detailed protocol to decellularize tissue sections while maintaining the sample attached to a glass slide. This protocol consists of consecutive washes and incubations of simple decellularizing agents: ultrapure water, sodium deoxycholate (SD) 2%, and deoxyribonuclease I solution 0.3 mg/mL (DNase I). This novel method has been optimized for a faster decellularization time (2–3 h) and a better correlation between dECM properties and native tissue-specific biomarkers, and has been tested in different types of tissues and species, obtaining similar results. Furthermore, this method can be used for scarce and valuable samples such as clinical biopsies.

0 Q&A 367 Views Nov 20, 2022

Actin filaments are essential for various biological activities in eukaryotic cellular processes. Available in vitro experimental data on these systems often lack details and information on sample preparation protocols and experimental techniques, leading to unreproducible results. Additionally, different experimental techniques and polymerization buffers provide different, sometimes contradictory results on the properties of these systems, making it substantially difficult to gather meaningful data and conclusive information from them. This article presents a robust, accurate, detailed polymerization protocol to prepare high-quality actin filament samples for light scattering experiments. It has been shown to provide unicity and consistency in preparing stable, dispersed, aggregates-free, homogenous actin filament samples that could benefit many other scientific research groups currently working in the field. To develop the protocol, we used conventional actin buffers in physiological conditions. However, it can easily be adapted to prepare samples using other buffers and biological fluids. This protocol yielded reproducible results on essential actin filament parameters such as the translational diffusion coefficient and electrophoretic mobility. Overall, suitable modifications of the proposed experimental method could generate accurate, reproducible light scattering results on other highly charged anionic filaments commonly found in biological cells (e.g., microtubules, DNAs, RNAs, or filamentous viruses).

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0 Q&A 498 Views Nov 5, 2022

Ubiquitination is a post-translational modification conserved across eukaryotic species. It contributes to a variety of regulatory pathways, including proteasomal degradation, DNA repair, and cellular differentiation. The ubiquitination of substrate proteins typically requires three ubiquitination enzymes: a ubiquitin-activating E1, a ubiquitin-conjugating E2, and an E3 ubiquitin ligase. Cooperation between E2s and E3s is required for substrate ubiquitination, but some ubiquitin-conjugating E2s are also able to catalyze by themselves the formation of free di-ubiquitin, independently or in cooperation with a ubiquitin E2 variant. Here, we describe a method for assessing (i) di-ubiquitin formation by an E1 together with an E2 and an E2 variant, and (ii) the cooperation of an E3 with an E1 and E2 (with or without the E2 variant). Reaction products are assessed using western blotting with one of two antibodies: the first detects all ubiquitin conjugates, while the second specifically recognizes K63-linked ubiquitin. This allows unambiguous identification of ubiquitinated species and assessment of whether K63 linkages are present. We have developed these methods for studying ubiquitination proteins of Leishmania mexicana, specifically the activities of the E2, UBC2, and the ubiquitin E2 variant UEV1, but we anticipate the assays to be applicable to other ubiquitination systems with UBC2/UEV1 orthologues.

0 Q&A 596 Views Oct 20, 2022

Infertility has become a major public health problem, with a male factor involved in about half the cases. Mice are the most widely used animal model in reproductive biology research laboratories, but changes in sperm parameters in mice can be subtle and, in the absence of official guidelines, it is important that analyses are carried out in a strict and reproductive manner. This protocol successively details the different steps required to obtain spermatozoa under good conditions, the measurement of sperm motility using a Computer Assisted Sperm Analysis System (CASA) device, the calculation of sperm concentration in the epididymides using a sperm counting cell, and the examination of sperm morphology. The combination of these assays provides an overview of the basic sperm parameters in mice. This is both a diagnostic and a decision-making tool for researchers to orient their scientific strategy according to the observed abnormalities.

0 Q&A 633 Views Oct 20, 2022

The human immunodeficiency virus 1 (HIV-1) consists of a viral membrane surrounding the conical capsid. The capsid is a protein container assembled from approximately 1,500 copies of the viral capsid protein (CA), functioning as a reaction and transport chamber for the viral genome after cell entry. Transmission electron microscopy (TEM) is a widely used technique for characterizing the ultrastructure of isolated viral capsids after removal of the viral membrane, which otherwise hinders negative staining of structures inside the viral particle for TEM. Here, we provide a protocol to permeabilize the membrane of HIV-1 particles using a pore-forming toxin for negative staining of capsids, which are stabilized with inositol hexakisphosphate to prevent premature capsid disassembly. This approach revealed the pleomorphic nature of capsids with a partially intact membrane surrounding them. The permeabilization strategy using pore-forming toxins can be readily applied to visualize the internal architecture of other enveloped viruses using TEM.

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0 Q&A 507 Views Oct 5, 2022

Cell bioprinting technologies aim to fabricate tissue-like constructs by delivering biomaterials layer-by-layer. Bioprinted constructs can reduce the use of animals in drug development and hold promise for addressing the shortage of organs for transplants. We recently introduced a laser-assisted drop-on-demand bioprinting technology termed Laser Induced Side Transfer (LIST). This technology can print delicate cell types, including primary neurons. This bioprinting protocol includes the following key steps: cell harvesting, bio-ink preparation, laser setup priming, printing, and post-printing analysis. This protocol includes a detailed description of the laser setup, which is a rather unusual setup for a biology lab. This should allow easy reproduction by readers with basic knowledge of optics. Although we have focused on neuron bioprinting, interested readers will be able to adapt the protocol to bioprint virtually any cell type.

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0 Q&A 742 Views Sep 20, 2022

The ex vivo experimentation with surgically discarded human skin represents a unique methodology amenable for mechanism and pharmacologic agent studies without the involvement of human subjects. Here, we describe a protocol that includes preparation, culture, and stimulation of human skin explants, and subsequent analyses by quantitative reverse transcription PCR and immunostaining. This protocol may also be applied for ex vivo studies of murine skin, reducing animal numbers and potentially harmful treatments. In our hands, this protocol has been used for wound healing, viral infection, and hair growth–related studies.

Graphical abstract:

Cartoon of explant skin culture.

Skin explant sits on top of a gelatin surgical sponge saturated with culture medium at an air–liquid interface.

0 Q&A 518 Views Sep 20, 2022

Cancer cells often overexpress glutaminase enzymes, in particular glutaminase C (GAC). GAC resides in the mitochondria and catalyzes the hydrolysis of glutamine to glutamate. High levels of GAC have been observed in aggressive cancers and the inhibition of its enzymatic activity has been shown to reduce their growth and survival. Numerous GAC inhibitors have been reported, the most heavily investigated being a class of compounds derived from the small molecule BPTES (bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide). X-ray structure determination under cryo-cooled conditions showed that the binding contacts for the different inhibitors were largely conserved despite their varying potencies. However, using the emerging technique serial room temperature crystallography, we were able to observe clear differences between the binding conformations of inhibitors. Here, we describe a step-by-step protocol for crystal handling, data collection, and data processing of GAC in complex with allosteric inhibitors using serial room temperature crystallography.

Graphical abstract:

Workflow for serial room temperature crystallography.

Diagram showing the processing and scaling routine for crystals analyzed using serial room temperature crystallography.

0 Q&A 1251 Views Aug 20, 2022

Abdominal surgeries are frequently associated with the development of post-surgical adhesions. These are irreversible fibrotic scar bands that appear between abdominal organs and the abdominal wall. Patients suffering from adhesions are at risk of severe complications, such as small bowel obstruction, chronic pelvic pain, or infertility. To date, no cure exists, and the understanding of underlying molecular mechanisms of adhesion formation is incomplete. The current paradigm largely relies on sterile injury mouse models. However, abdominal surgeries in human patients are rarely completely sterile procedures. Here, we describe a modular surgical procedure for simultaneous or separate induction of sterile injury and microbial contamination. Combined, these insults synergistically lead to adhesion formation in the mouse peritoneal cavity. Surgical trauma is confined to a localized sterile injury of the peritoneum. Microbial contamination of the peritoneal cavity is induced by a limited perforation of the microbe-rich large intestine or by injection of fecal content. The presented protocol extends previous injury-based adhesion models by an additional insult through microbial contamination, which may more adequately model the clinical context of abdominal surgery.

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